Human Induced Pluripotent Stem (IPS) Cells
In 2006, Japanese Yamanaka laboratory successfully obtained a pluripotent stem cell, which was very similar to mouse embryonic stem cells in characteristics, by transducing four transcription factors (KLF4,c-Myc, SOX2, OCT-4) into mouse embryonic stem cells and adult fibroblast through retrovirus. Soon, using the same method, human fibroblast was transduced to induce human pluripotent stem cells successfully. Afterwards, a variety of IPS cells induced from patient cells with hereditary disease were obtained successfully. IPS cells are similar to embryonic stem cells in characteristics. The embryonic stem cells can differentiate into all types of somatic cells, which are able to be used to repair the tissue injury from diseases or hurt. So the embryonic stem cells have a very extensive application prospect in the field of regenerative medicine. However, application of embryonic stem cells in medicine has two important obstacles: one is immunologic rejection after transplantation, the other is the ethical consideration of using a human embryo. Even if the embryonic stem cells were obtained through somatic cell nuclear transfer, there are still ethical problems. Nevertheless, the human IPS cells can be obtained from patient cells, which does not have the problem of immunologic rejection. And since no human embryo is destroyed or human ootids are used, the ethical problem of using embryonic stem cells does not exist. These advantages above enable the IPS technology to have a better application prospect in regenerative medicine.
The Technology of Inducing Pluripotent Stem (IPS) Cells
Initially, inducing Pluripotent Stem cells needed to use replication-deficient retrovirus or Lentiviral Vector, which could transduce the reprogramming factors into cells. These viral vectors would integrate into genome of the host cell. Although these exogenous genes are silent in IPS cells at most cases, once being reactivated, they will induce tumor. Leaked expression of these genes also possibly enables IPS cells to differentiate and ripe incompletely, resulting in the increasing risk of forming immaturity teratoma. Viral integration also possibly activates or terminates expression of endogenous genes. In the history of gene therapy, using the technology of retrovirus integration resulted in leukemia because of activating oncogenes. Many laboratories tried to use the technology of non-viral integration to induce Pluripotent Stem cells. Adenovirus and/or plasmid were used as vectors to introduce reprogramming factors into cells, thus obtained IPS cells successfully. But the rate of obtaining IPS cells was very low. OriP/EBNA-1 plasmid episome was used as vector to induce IPS cells. Some laboratories used Cre/loxp, transposon/transposase, to remove the exogenous gene being integrated into genome after obtaining IPS cells. Another method to avoid integration of exogenous genes was to replace reprogramming factors by chemicals. So far, from the results published, no completely alternative reprogramming transcription chemicals or combination has been found, only one or two reprogramming factors could be replaced. Even though no foreign gene was integrated into IPS cells, the method mentioned above could not completely avoid genomic change, for instance, the method of plasmid transformation would bring genomic integration with low chance, while chemicals could lead to gene mutation.
Protein Transduction
Initially, protein transduction was originated from the research of HIV TAT protein. People found that the entire HIV TAT protein could enter cells to activate transduction of viral gene. Further studies showed that a region (TAT PTD) of HIV TAT protein was responsible for the function of entering cells. Coupling or fusion of TAT PTD and macromolecules was found useful for the macromolecules to enter cell. Study showed arginine with positive charges was necessary for TAT PTD to enter cytoplasm through cell membrane. Any mutation of an arginine would lead to loss of the transduction function. Based on this, polyarginine was found to possess the function of transduction similarly. The technology of protein transduction by TAT PTD or the other protein transducing peptide has a great potential for macromolecular pharmaceuticals to enter cells and exert their function.
Cleavage of SUMO Fusion Proteins
Small ubiquitin-like modifier (SUMO) could covalently modify protein. SUMO modification can regulate various cell process including nuclear transfer, signal transduction and stability of protein. Ulp1, a SUMO protease, could specifically recognize the tertiary structure of SUMO, and cleave at the joint of SUMO and its modified protein. When SUMO fusing with other protein (equivalently SUMO modifies N-terminal amino of target protein), Ulp1 could specifically remove SUMO, and release the target protein completely.
Protein Inducing Pluripotent Stem Cells
The reprogramming factors (KLF4, c-Myc, SOX2, OCT-4) were introduced into cells by the technology of protein transduction, which can avoid the security problems mentioned above. A laboratory of Scripps institute successfully obtained mouse IPS cells by a recombinant protein of four reprogramming factors expressed in E. coli, with the addition of a kind of chemical (HDAC inhibitor). The recombinant reprogramming factors used in the laboratory were OCT-4, KLF4, Sox2, c-Myc. The four reprogramming factors were introduced into cells by polyarginine with C-terminal fusion. The technology can completely overcome all disadvantages when the reprogramming factors were introduced into cells by using DNA, thus enables the possible application of IPS cells in Regenerative Medicine to take a significant step forward. However, the protein transduction peptide—polyarginine fused the reprogramming factors directly. Since the transduction peptide carried strong positive charges, which possibly nonspecifically bind with genomic DNA carrying negative charges, thus the transcription factor fused with transduction peptide could nonspecifically change gene expression. The nonspecific change of gene expression could lead to low rate of inducing Pluripotent Stem Cells or some permanent change of gene expression, thus influenced subsequent differentiation and maturity of the IPS cells. Hence, obtaining a preparation, which could overcome the disadvantages above in inducing Pluripotent Stem Cells, had a great significance for human IPS cells to use practically in Regenerative Medicine.